Monitoring Intestinal Organoid–Derived Monolayer Barrier Functions with Electric Cell–Substrate Impedance Sensing (ECIS)

The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro. So far, the ECIS technology has exclusively been performed on cell lines. Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids. Key features • We present a protocol that allows the assessment of various cell functions, such as proliferation and barrier formation, with ECIS on organoid-derived monolayers. • The protocol facilitates intestinal barrier research on patient tissue-derived organoids, providing a valuable tool for disease modeling.

This protocol is used in: Sci Rep (2023), DOI: 10.1038/s41598-023-45160-w The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity.Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro.So far, the ECIS technology has exclusively been performed on cell lines.Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases.In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids.

Background
The intestinal epithelium is represented as a cellular monolayer that separates the luminal content from the rest of the body [1].Apart from enabling digestion and absorption of food, it serves as a crucial barrier for warding off potentially pathogenic microbes [1].Damage and impairment of the gut barrier are observed through the course of various intestinal diseases such as necrotizing enterocolitis and inflammatory bowel disease [2,3].A major boost to the in vitro modeling of the intestinal epithelial barrier came with the emergence of organoid technology.Organoids are self-organizing, three-dimensional (3D) structures that are grown in vitro from stem cells [4].They recapitulate many structural and functional aspects of their parent organ more accurately than the commonly used two-dimensional (2D) cell lines [4].Therefore, organoids have become a frequently used model among researchers.Since it remains challenging to study epithelial barrier formation in 3D structures, several studies cultured the intestinal organoids as a monolayer in a Transwell system [1].The Transwell system is the most commonly used in vitro model for intestinal barrier research [5,6].It consists of a porous cell culture insert that can be placed in a traditional cell culture well plate [5].The gut epithelial cells are grown on the permeable membrane of the insert to create a cell monolayer with a luminal and basolateral compartment.Evaluation of barrier formation is assessed by measuring the transepithelial electrical resistance (TEER) with electrodes placed on either side of the membrane [5,6].While measuring TEER with the typical handheld chopstick electrode epithelial voltohmmeter (EVOM) device remains the gold standard for assessing the integrity of barrier models, this technique requires removal of the cultures from the incubator to test each well individually.Such approach might disrupt the cell layers and, in addition, variations in electrode positioning could hinder reproducibility of measurement because of non-uniform electric field created by the chopstick electrodes [6].An alternative to EVOM is electrical cell-substrate impedance sensing (ECIS).ECIS uses the same principle as the EVOM, except that the electrodes are integrated into the bottom of the well on which the cells are grown [6].The presence of the electrodes in the wells allows cells to attach and proliferate directly on top of the electrodes, resulting in more localized and sensitive impedance measurements of the cell barrier.The ECIS enables impedance measurements at a broad scale of electrical frequencies, ranging from 62.5 Hz to 64 kHz.In addition, depending on the electrode placement and size, the ECIS can be used to determine additional properties of the epithelial cell layer such as cell attachment, migration, and proliferation [6].Studying cell attachment, proliferation, and spreading yields insights into fundamental cellular behaviors, enhancing our understanding of basic biological processes in health and disease.Apart from monitoring cell barrier formation, the cells can be subjected to (lethal) electrical currents to inflict cellular damage [7].The ECIS is therefore a potentially interesting cell-based system to study gut barrier formation and to screen for drugs capable of resolving cell damage and achieving higher mucosal healing rates.Here, we show that the ECIS technology can be successfully applied on gut organoid 2D monolayers.

Human IntestiCult TM organoid growth medium
Combine 50 mL of IntestiCult TM OGM human basal medium with 50 mL of organoid supplement and 1 mL of Pen/strep.

Human IntestiCult TM organoid differentiation medium
Combine 50 mL of IntestiCult TM ODM human basal medium with 50 mL of organoid supplement and 1 mL of Pen/strep.

Coating solutions
1. 100 mM L-cysteine Dissolve 12.12 mg of L-cysteine in 1 mL of Milli-Q water.Pass the solution through a 0.2 µm syringe sterilization filter.The solution can be kept in the fridge for up to two weeks.Dilute the stock solution 10× in sterile Milli-Q water before the start of the experiment.

0.1% acetic acid solution
Add 25 µL of acetic acid to 25 mL of Milli-Q water.Pass the solution through a 0.2 µm syringe sterilization filter.

Collagen/0.1% acetic acid solution
Wells should be coated with 10 µg collagen/cm

Procedure
Conduct all cell and array manipulations within a sterile environment using a laminar flow cabinet.

A. Preparation of the 16-well station
1. Place the ECIS 16-well station in an incubator at 37 °C at least one day before the experiment to prewarm the array holder (Figure 1A). 2. Add water to the incubator's water reservoir to prevent the wells from drying out.

B. Preparation of the arrays
The choice of array depends significantly on the specific scientific question and the type of cells involved.5. Resuspend the cells vigorously for 15-20× with a p1000 pipette to generate a single-cell suspension.Adding a 200 µL tip on top of the 1,000 µL tip greatly improves the mechanical disruption of the organoids.No cell clumps or organoid structures should be visible with the naked eye.See general note 7. 6. Inactivate the TrypLE with 10 mL of Ad-DF+++.7. Spin the cells at 340× g for 5 min at 4 °C.Remove the supernatant.8. Resuspend the cells in organoid growth medium (OGM) supplemented with ROCK-inhibitor (1:1,000) and perform a cell count.9. Prepare the single-cell suspension in the range of 150,000-300,000 cells per well.Adjust the volume with OGM supplemented with ROCK-inhibitor.10.Pause the baseline measurements and take the array out of the incubator (Figure 2E).11.Remove the media in the array.12. Add 400 µL of the homogenized cell suspension to each well.Do not forget to include an empty control.13.Put the array back in the 16-well station and resume the measurements (Figure 2E).See general note 8.

E. Experiment run and measurements
The frequency-selection ECIS experiments rely on the research goal and the characteristics of the cells under investigation.In resistance analysis, the 4,000 Hz frequency is used as the default setting, as this frequency is regularly used due to its relevance in capturing subtle changes associated with cell behavior and barrier function, particularly in the context of tight junction formation and integrity.However, the optimal frequency may vary, depending on the cell type and the specific experimental conditions.The frequency scan performed with the ECIS software on fetal and adult intestinal organoids showed that capacitance measurements should be analyzed at 64 kHz, whereas resistance should be analyzed at 500 Hz (Figure 3A, 3B).The capacitance and resistance data shown in this protocol paper are therefore measurements performed at a frequency of 64 kHz and 500 Hz, respectively.

Figure 3. Optimal alternating current (AC) frequencies for analysis of intestinal organoid monolayers.
The frequency displaying the greatest difference between cell-free (empty) and cell-covered conditions is deemed ideal for subsequent analysis.A. Capacitance data from fetal organoids grown on organoid growth medium (OGM), displayed as cell-to-cell-free ratios, exhibits a maximum response at 64 kHz (dotted line).B. However, 500 Hz (dotted line) or 1 kHz seem to be the optimal frequencies for analysis of resistance data.
Compared to cell lines, much higher organoid cells numbers are needed to reach confluence shortly after seeding.As an example, approximately 50,000 CaCo2 cells are needed to obtain a monolayer within 24 h.ECIS Published: Mar 05, 2024 capacitance measurement, which indicates electrode coverage, shows that if you seed two different organoid cell numbers per well, 150,000 or 300,000, no monolayers are formed within the first 30 h after seeding (Figure 4A-4C).After approximately 60 h, a monolayer has formed in the wells with 300,000 cells per well, and an additional 30 h is needed before a monolayer has formed in the wells with 150,000 cells per well (Figure 4D-4E).ECIS resistance measurements (500 Hz) correlate inversely with the capacitance measurements, showing that the transepithelial resistance is increasing while the monolayer is forming (Figure 4F-4J).Approximately 300,000 cells per well are needed for human intestinal organoids to reach confluence within three days, but this could differ slightly per donor depending in its growing rate.Perform a pilot experiment to determine the optimal cell number that fits your research question.or triangle shaped).A. As cells proliferate and cover the electrode surfaces, electrical current is impeded.The capacitance measured during cell proliferation is inversely correlated with surface coverage until a complete monolayer has formed.B-E.The capacitance measured at 64 kHz (indicative of electrode coverage) indicates that a full monolayer was achieved after 60 and 90 h when 150,000 (n = 12) or 300,000 (n = 3) cells were seeded, respectively.F. A significant increase in resistance is observed as the monolayers form.G-I.Resistance continues to increase and reaches a plateau at 4,000 Ω when measured at 500 Hz (indicating intact cell barrier).Bars represent mean ± SD.P < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****) as determined with an unpaired t-test.

F. Changing the medium during an experiment
1. Click on Pause to halt data acquisition.The experimental clock will continue to run. 2. Take the array out of the holder and change the medium under a laminar flow bench.Change the medium to medium without ROCK-inhibitor 2-3 days after seeding and continue to refresh medium every 2-4 days.See general note 9. 3.Return the array to the holder and click Check to check if arrays were placed back correctly.4. Wait for 5-10 min before resuming the measurements, as temperature changes affect the measurements.5. Click Resume to restart data acquisition.The measurement software will include a time mark in the data set.See general note 10.

G. Differentiation of cells
If the research question requires the cells to reflect the mature intestine and the monolayer to contain all major cell types present within tissues, an additional differentiation step might be performed [8].By switching the culture medium from OGM to organoid differentiation medium (ODM) after the gut monolayer has been formed, an additional increase in the transepithelial resistance is observed (Figure 5).Gut cells cultured in ODM contain physiologically relevant properties of differentiated cells, such as nutrient absorption and expression of digestive enzymes and tight junction proteins.

H. Wound-healing assay
Damage to the intestinal epithelial barrier is observed in a number of gut diseases.Once a stable monolayer has formed, the ECIS can be used to inflict cellular damage by subjecting the cells to a lethal electrical current.The electrical current specifically kills the cells on top of the small gold electrodes, which subsequently die and detach from the electrode, creating a wound that is healed by neighboring cells that have not been submitted to the current [7].This function allows for the screening of drugs that might provide significant clinical benefit by improved or faster wound healing.Determining the appropriate settings for the specific cell type is crucial for electrical wounding.If the wounding duration is too brief, it may lead to inadequate cell removal, while excessively long or harsh wounding can potentially damage the electrodes.For the wound-healing assays, 8W10E confluent intestinal epithelial monolayers derived from organoids were subjected to different currents.While applying a current of 4,000 μA and a frequency of 50,000 Hz for 30 s is capable of inducing enough cell damage for the cells to break their tight junction, the capacitance data shows that the current is not strong enough to kill all the cells present on the electrode.However, successful wounding on organoid-derived monolayers is achieved after applying a current of 5,000 μA and a frequency of 50,000 Hz for 60 s (Figure 6).

Figure 1 .
Figure 1.Electrical cell-substrate impedance sensing (ECIS) setup.A. The 16-well station is placed in the incubator at 37 °C.B. The ECIS ® Z-Theta instrument, including laptop, is placed in close proximity to the incubator holding the 16-well station.C. The 8W test array is used to calibrate the ECIS.Running the test array should provide measurements with values (close-up picture) mentioned on the array.D. The 8W ECIS arrays

Figure 2 .
Figure 2. Electrical cell-substrate impedance sensing (ECIS) software.A. Upon opening the ECIS software, click Setup (black arrow) in the right window to measure the impedance of the device and detect an open circuit.B. Arrays/wells that have been properly connected are indicated in green in the left-bottom window of the screen, whereas arrays or wells with an open circuit are shown in red.C. Readjust the placement of the array and select Setup (black arrow) again to confirm correct placement of the array.By selecting check (button underneath Setup), measurements of the test array will be made using the default frequency of 4,000 Hz.If electrodes were not properly stabilized after the L-cysteine treatment, an additional stabilization step can be performed with the ECIS by selecting stabilize (green arrow).The same right pane holds options for the acquisition mode (outlined with a black square), the Wound/Electroporate Setup function (outlined with a red square), and the Start (red arrow) button.D. Measurements performed by selecting Check, e.g., results from the test array, are shown on a pop-up screen.E. Data acquisition is

Figure 4 . 9 Published
Figure 4. Barrier formation in 2D intestinal organoid-derived monolayers.Intestinal organoid cells from the same fetal tissue culture were seeded in two different ECIS 8W10E arrays (indicated in the graph as round

Figure 5 .
Figure 5. Organoid cell differentiation results in increased resistance values.Intestinal organoid cells from adult tissue were seeded into 8W10E ECIS arrays and cultured in IntestiCult organoid growth medium (OGM).After 118 h, when the capacitance plateau phase was reached but resistance values were still increasing

Figure 6 .
Figure 6.Wounding of 2D organoid monolayers.Monolayers formed by adult organoids were grown in organoid growth medium (OGM).A. Applying a current of 4,000 μA and a frequency of 50,000 Hz for 30 s